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Revision as of 12:59, 19 September 2018

PROJECT DESCRIPTION

About

Scientists all over the world in different fields are using microorganisms for their research - that is nothing new. But most of them are using monocultures and therefore miss thousands of possibilities which arise in co-cultures. It is estimated that less than 1% of all microorganisms have been successfully cultivated1. A reason for that might be the very artificial conditions in the laboratory. In nature no organism is completely isolated, but rather lives in very complex systems. Scientist have only just begun to investigate the complex interaction between different microorganisms but yet often fail at the cultivation. Easily obtainable and stable co-cultures would allow research of yet uninvestigated species and might give further insight into cell-cell-interactions between microorganisms2. One problem of isolated cultures is the regular use of antibiotics as selective pressure and prevention of contaminations which leads towards multiple resistances. Here as well a co-culture with nutrient dependencies instead of higher antibiotic concentration is a better alternative. Pharmacotherapeutic companies spend a lot of money on developing new antibiotics, while in co-culturing conditions some microorganisms show differential gene expression and might produce new antibiotics on their own3. In general, co-cultures might provide a great opportunity as a new method for cultivation and the production of new antibiotics or industrially interesting products. There is yet a lot of potential for new co-cultures and we - Team iGEM 2018 HHU - want to tackle that challenge.


The Challenges

Before starting research, first the safety must be ensured. But this is a self solving problem, since the organisms are only able to live in a community that is designed by the researcher itself. That includes a detailed control of the microorganisms, the cell density, their behavior and environmental conditions. For industrial usage the production must be scalable and high throughput must be guaranteed as well. Furthermore, general technologies for a normed use for research projects that are also easily applicable for large scale production must be achieved4. One goal would be the reduction and simplification of processing steps and easy-to-use protocols for stable co-cultures to make cultivation and production faster and cheaper. Other requirements for making co-cultures a profitable alternative to isolated culturing are big product diversity and efficient extraction of them. For scientists the possibility to build complex systems and methods for big data collection are desirable and highly demanded.


The Project

Since our co-cultures should be universally usable for laboratories all over the world, we plan to design it as easy and modular as possible. Therefore, the Golden Gate modular cloning standard techniques (MoClo) were used besides Gibson Assembly for our cloning steps, which allows exchange of all parts to adjust the co-culture to every possible condition5,6. We envision a standardised system, a three-way co-culture with a selection of organisms and regulations. `Trinity` is going to incorporate different dependencies between the organisms in order to control their growth and enable them to adjust each others behaviour. Project Trinity is divided into three systems. Our system 1 deals with the dependence of the organisms Escherichia coli, Saccharomyces cerevisiae and Synechococcus elongatus sp. PCC 7942 based on the availability of essential nutrients such as nitrogen7, phosphate8 and carbon9. The aim of system 2 is the dependency of E.coli, S. cerevisiae and S. elongatus , achieved through the exploitation of auxotrophies. S. cerevisiae is in our case auxotrophic for lysine which is produced by E.coli10(. At the same time, E.coli has an auxotrophy for leucine, which, in turn, is provided by S. cerevisiae. S. elongatus shows no auxotrophy and enriches the media with the monosaccharides glucose and fructose. This way E.coli and S. cerevisiae are regulating each other and do not overgrow S. elongatus. Each organism is responsible for the production of one substance, amino acid or glucose, and thus makes the other organism dependent on it. System 3 is utilising the quorum sensing mechanism to regulate cell density in the population by using bacterial communication molecules. E.coli regulates its own cell density by expressing a lysis gene after induction by AHL1 quorum sensing molecules11. For S. cerevisiae two different systems were introduced. The first one utilises the fact that upon recognition of the complementary mating type factor pheromone (MAT `a` for `alpha` yeast) yeast cells go into a cell cycle arrest and stop growing12. The second system depends on the design of a synthetic promoter that activates another lysis gene after the recognition of AHL2 molecules13. The following box provides a graphical overview as well as a summery of the role of each organism in the system for each Level: .



Our Trinity

Since our co-cultures should be universally usable for laboratories all over the world, we plan to design it as easy and modular as possible. Therefore, the Golden Gate modular cloning standard techniques (MoClo) were used besides Gibson Assembly for our cloning steps, which allows exchange of all parts to adjust the co-culture to every possible condition5,6. We envision a standardised system, a three-way co-culture with a selection of organisms and regulations. `Trinity` is going to incorporate different dependencies between the organisms in order to control their growth and enable them to adjust each others behaviour. Project Trinity is divided into three systems. Our system 1 deals with the dependence of the organisms Escherichia coli, Saccharomyces cerevisiae and Synechococcus elongatus sp. PCC 7942 based on the availability of essential nutrients such as nitrogen7, phosphate8 and carbon9. The aim of system 2 is the dependency of E.coli, S. cerevisiae and S. elongatus , achieved through the exploitation of auxotrophies. S. cerevisiae is in our case auxotrophic for lysine which is produced by E.coli10. At the same time, E.coli has an auxotrophy for leucine, which, in turn, is provided by S. cerevisiae. S. elongatus shows no auxotrophy and enriches the media with the monosaccharides glucose and fructose. This way E.coli and S. cerevisiae are regulating each other and do not overgrow S. elongatus. Each organism is responsible for the production of one substance, amino acid or glucose, and thus makes the other organism dependent on it. System 3 is utilising the quorum sensing mechanism to regulate cell density in the population by using bacterial communication molecules. E.coli regulates its own cell density by expressing a lysis gene after induction by AHL1 quorum sensing molecules11. For S. cerevisiae two different systems were introduced. The first one utilises the fact that upon recognition of the complementary mating type factor pheromone (MAT `a` for `alpha` yeast) yeast cells go into a cell cycle arrest and stop growing12. The second system depends on the design of a synthetic promoter that activates another lysis gene after the recognition of AHL2 molecules13.

References

  1. Katz, Micah, Bradley M. Hover, and Sean F. Brady. "Culture-independent discovery of natural products from soil metagenomes." Journal of industrial microbiology & biotechnology 43.2-3 (2016): 129-141.
  2. Goers, Lisa, Paul Freemont, and Karen M. Polizzi. "Co-culture systems and technologies: taking synthetic biology to the next level." Journal of The Royal Society Interface 11.96 (2014): 20140065.
  3. Adnani, Navid, et al. "Coculture of Marine Invertebrate-Associated Bacteria and Interdisciplinary Technologies Enable Biosynthesis and Discovery of a New Antibiotic, Keyicin." ACS chemical biology 12.12 (2017): 3093-3102.
  4. Padmaperuma, Gloria, et al. "Microbial consortia: a critical look at microalgae co-cultures for enhanced biomanufacturing." Critical reviews in biotechnology 38.5 (2018): 690-703.
  5. Lee, Michael E., et al. "A highly characterized yeast toolkit for modular, multipart assembly." ACS synthetic biology 4.9 (2015): 975-986.
  6. Iverson, Sonya V., et al. "CIDAR MoClo: improved MoClo assembly standard and new E. coli part library enable rapid combinatorial design for synthetic and traditional biology." ACS synthetic biology 5.1 (2015): 99-103.
  7. Shaw, A. Joe, et al. "Metabolic engineering of microbial competitive advantage for industrial fermentation processes." Science 353.6299 (2016): 583-586.
  8. Kanda, Keisuke, et al. "Application of a phosphite dehydrogenase gene as a novel dominant selection marker for yeasts." Journal of biotechnology 182 (2014): 68-73.
  9. Niederholtmeyer, Henrike, et al. "Engineering cyanobacteria to synthesize and export hydrophilic products." Applied and environmental microbiology 76.11 (2010): 3462-3466.
  10. Schrumpf, Barbel, et al. "A functionally split pathway for lysine synthesis in Corynebacterium glutamicium." Journal of bacteriology 173.14 (1991): 4510-4516.
  11. Dong, Yi-Hu, et al. "Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase." Nature411.6839 (2001): 813.
  12. Williams, Thomas C., Lars K. Nielsen, and Claudia E. Vickers. "Engineered quorum sensing using pheromone-mediated cell-to-cell communication in Saccharomyces cerevisiae." ACS synthetic biology 2.3 (2013): 136-149.
  13. Scott, Spencer R., et al. "A stabilized microbial ecosystem of self-limiting bacteria using synthetic quorum-regulated lysis." Nature microbiology 2.8 (2017): 17083.


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